Biochemical and Biophysical Research Communications 394 (2010) 728–732

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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

The citrus ﬂavonoids hesperetin and naringenin block the lipolytic actionsof TNF-a in mouse adipocytes
Hiroki Yoshida a,*, Norito Takamura a, Tsuyoshi Shuto b, Kenji Ogata a, Jin Tokunaga a, Keiichi Kawai c,
Hirofumi Kai babc

Second Department of Clinical Pharmacy, School of Pharmaceutical Sciences, Kyushu University of Health and Welfare, 1714-1 Yoshino, Nobeoka City, Miyazaki 882-8508, Japan
Department of Molecular Medicine, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oehonmachi, Kumamoto 862-0973, Japan
Division of Health Sciences, Graduate School of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa 920-0942, Japana r t i c l e

i n f oArticle history:
Received 8 March 2010
Available online 15 March 2010
Keywords:
Hesperetin
Naringenin
TNF-a
Lipolysis
Free fatty acid
Adipocyte
Flavonoida b s t r a c t
Obese adipose tissue is characterized by an excessive production of inﬂammatory adipokines including
tumor necrosis factor-a (TNF-a). TNF-a stimulates free fatty acid (FFA) secretion through adipocyte lipolysis, and increased plasma levels of FFA promote insulin resistance. In this report, we show that hesperetin and naringenin, two citrus ﬂavonoids, inhibit TNF-a-stimulated FFA secretion from mouse adipocytes.
These ﬂavonoids block the TNF-a-induced activation of the NF-jB and ERK pathways. Moreover, hesperetin and naringenin prevent TNF-a from downregulating the transcription of two antilipolytic genes, perilipin and PDE3B. These effects are mediated through the inhibition of the ERK pathway. In contrast, theinhibition of the NF-jB pathway by hesperetin and naringenin suppresses the transcription of IL-6, which
induces FFA secretion in an autocrine manner. Our results provide novel evidence that hesperetin andnaringenin directly inhibit TNF-a-stimulated FFA secretion. These ﬁndings may be useful for ameliorating
FFA-induced insulin resistance.
Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction
Obese adipose tissue produces excess inﬂammatory adipokines,
including tumor necrosis factor-a (TNF-a), which are involved in
the development of insulin resistance and type 2 diabetes [1,2].
TNF-a plays a pivotal role in obesity-related insulin resistance,
and its expression is increased in the obese adipose tissue of bothrodents and humans [3–5]. TNF-a induces insulin resistancethrough its direct ability to attenuate insulin receptor signaling[6,7]. Additionally, several reports suggest that TNF-a indirectly
promotes insulin resistance by increasing the circulating levels of
free fatty acid (FFA) [8–10]. Interestingly, the interstitial levels of
TNF-a and FFA in adipose tissue are positively correlated [11].
Chronically elevated plasma levels of FFA promote insulin resistance by impairing peripheral glucose utilization [10,12]. Thus,
the reduction of excess FFA levels is important in the improvementof insulin resistance.
TNF-a promotes FFA secretion through adipocyte lipolysis.
Although the mechanism by which TNF-a induces lipolysis has
Abbreviations: TNF-a, tumor necrosis factor-a; FFA, free fatty acid; NF-jB,
nuclear factor-jB; ERK, extracellular signal-regulated kinase; PDE3B, phosphodiesterase-3B; IL-6, interleukin-6; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
* Corresponding author. Fax: +81 982 23 5539.
E-mail address: h-yoshida@phoenix.ac.jp (H. Yoshida).
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.03.060

yet to be completely elucidated, studies have proposed that TNFa downregulates the expression of antilipolytic genes, such as perilipin and phosphodiesterase-3B (PDE3B) [3,13,14]. Perilipin is located on the surface of intracellular triglyceride lipid droplets
and regulates the access of hormone-sensitive lipase (HSL), whichhydrolyzes triglycerides to FFA and glycerol [15–17]. PDE3B is the
major hydrolytic enzyme of cAMP activated by insulin signaling[18,19]. Decreased intracellular levels of cAMP lead to reduced
activity of protein kinase A (PKA), which phosphorylates and activates HSL [20]. Some intracellular signaling pathways mediate theeffects of TNF-a on adipocyte lipolysis. Nuclear factor-jB (NF-jB)
and extracellular signal-regulated kinase (ERK) are important for
TNF-a-induced lipolysis, because they regulate the expression of
lipolysis-related genes including perilipin and PDE3B [21–23].
Thus, the regulation of antilipolytic gene expression through theactivation of intracellular signaling pathways may be the mechanism by which TNF-a induces lipolysis.
Flavonoids, which are found in fruits and vegetables, have beneﬁcial effects on health. Hesperetin and naringenin are ﬂavonoids
that are abundant in citrus fruits, and they exert antioxidant, antiinﬂammatory and antiproliferative effects [24–27]. A cohort study
found that the intake of hesperetin and naringenin reduces the riskof chronic diseases such as cerebrovascular disease and asthma [28].
However, the effects of hesperetin and naringenin on adipocyte
function, especially FFA secretion, remain largely unknown.

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In this report, we show that hesperetin and naringenin block
TNF-a-stimulated FFA secretion by inhibiting the NF-jB and ERK
pathways in mouse adipocytes. The inhibition of the ERK pathway
prevents TNF-a from downregulating the transcription of two antilipolytic genes, perilipin and PDE3B. In contrast, the inhibition of
the NF-jB pathway suppresses the transcription of IL-6, which also
induces FFA secretion. Our results provide novel evidence that hesperetin and naringenin directly inhibit TNF-a-stimulated FFA
secretion. These ﬁndings may be useful for developing treatments
to ameliorate FFA-induced insulin resistance.
2. Materials and methods
2.1. Reagents
We purchased the following reagents: TNF-a, IL-6, control IgG
and IL-6 neutralizing antibody (R&D Systems; Minneapolis, MN);
hesperetin (Wako Pure Chemical Industries; Osaka, Japan);naringenin, isobutylmethylxanthine (IBMX) and dexamethasone(DEX) (Sigma; St. Louis, MO); insulin (Cell Science & Technology
Institute; Sendai, Japan); Dulbecco’s modiﬁed Eagle’s medium(DMEM), bovine serum (BS) and fetal bovine serum (FBS) (Invitrogen; Carlsbad, CA); MG-132 (BIOMOL; Plymouth Meeting, PA); andU0126 (Cell Signaling Technology; Beverly, MA).
2.2. Cell culture
3T3-L1 cells (Health Science Research Resources Bank; Osaka,
Japan) were maintained in DMEM supplemented with 10% BS.
After the preadipocytes reached conﬂuency, they were induced
to differentiate into mature adipocytes by replacing the medium
with 10% FBS-supplemented DMEM containing 0.5 mM IBMX,
0.25 lM DEX and 5 lg/mL insulin for 3 days. Then, the medium
was replaced with 10% FBS-supplemented DMEM containing
5 lg/mL insulin, and it was changed every 3 days for the next
6 days. Primary adipose cells derived from mouse epididymal fat
pads (Primary Cell; Hokkaido, Japan) were cultured according to
the manufacturer’s instructions.
2.3. Fatty acid assay
Differentiated adipocytes were incubated overnight in DMEM
with 2.0% fatty acid-free bovine serum albumin (Sigma) in the absence of serum. The following morning, cells were treated with
TNF-a or IL-6 for 24 h after a 30-min pretreatment with ﬂavonoids,
signaling inhibitors, or neutralizing antibodies. The levels of freefatty acids were determined by using an acyl-CoA oxidase-based
colorimetric kit (NEFA-V2; Alfresa, Osaka, Japan).
2.4. Cell viability assay
Viability was assessed by using an MTT assay kit (BioAssay Systems; Hayward, CA), according to the manufacturer’s instructions.
This assay is based on the conversion of a tetrazolium salt, MTT, toformazan by living cells. In brief, cells were incubated with MTT reagent for 4 h at 37 °C, and the produced formazan was dissolved
with solubilization solution. Absorbance was measured at
570 nm, and data from TNF-a-treated wells were expressed relative to control wells to obtain a cell survival index.
2.5. Real-time RT-PCR analysis
Total RNA was isolated (TRIzol reagent; Invitrogen), and the reverse transcription reaction was performed (Transcriptor First

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Strand cDNA Synthesis Kit; Roche, Basel, Switzerland), according
to the manufacturers’ instructions. In brief, the reverse transcription reaction was performed with oligo(dT) and random hexamersfor 10 min at 25 °C, followed by 60 min at 50 °C. PCR ampliﬁcation
was performed by using LightCycler FastStart DNA Master PlusSYBR Green 1 (Roche). Each reaction contained 4 lL of 5 Master
Mix, 1 lL of template cDNA and 500 nM primers in a ﬁnal volumeof 20 lL. Duplicate samples were incubated in the LightCycler for
an initial denaturation at 94 °C for 30 s, followed by 40 PCR ampliﬁcation cycles of 95 °C for 10 s, 60 °C for 5 s and 72 °C for 12 s. To
conﬁrm the ampliﬁcation of speciﬁc transcripts, melting curve proﬁles were produced at the end of each PCR. The relative quantity ofmRNAs was determined by using the comparative Ct method andnormalized by using b-actin as an endogenous control. All samples
were run in duplicate. The primers were as follows: perilipin forward, 50 -CTC TGG GAA GCA TCG AGA AG-30 ; perilipin reverse, 50 GCA TGG TGT GTC GAG AAA GA-30 ; PDE3B forward, 50 -GGT GAT
GGT GGT GAA GAA-30 ; PDE3B reverse, 50 -AGT GAG GTG GTG CAT
TAG-30 ; IL-6 forward, 50 -AGT TGC CTT CTT GGG ACT GAT-30 ; IL-6
reverse, 50 -TCC ACG ATT TCC CAG AGA AC-30 ; b-actin forward, 50 AGC CAT GTA CGT AGC CAT CC-30 ; and b-actin reverse, 50 -TCC
CTC TCA GCT GTG GTG GTG AA-30 .
2.6. Western blot analysis
Cells were lysed with RIPA buffer containing PMSF, proteasome
inhibitor cocktail and sodium orthovanadate (Santa Cruz Biotechnology; Santa Cruz, CA). Whole cell extracts were separated on
10% SDS–PAGE gels and transferred to polyvinylidene diﬂuoride
membranes (Bio-Rad Laboratories; Hercules, CA). The membranes
were blocked with a TBS solution containing 0.1% Tween 20(TBS-T) and 2% Blocking Agent (GE Healthcare UK Ltd; Buckinghamshire, UK). After three washes in TBS-T, the membranes wereincubated with a 1:2000 dilution of a primary antibody. After another three washes in TBS-T, the membranes were incubated witha 1:10,000 dilution of the corresponding secondary antibody.
Chemiluminescence reagent ECL Advance (GE Healthcare) wasused to visualize the blots. Antibodies against IjB-a, phosphoERK1/2 (Thr202/Tyr204) and b-actin were purchased from Cell Signaling Technology.
2.7. Statistical analysis
Values are given as means ± SD. Data were considered signiﬁcant at p < 0.01 based on ANOVA with Student–Newman–Keuls(SNK) post-hoc test.

3. Results
3.1. Hesperetin and naringenin inhibit TNF-a-stimulated FFA secretionfrom mouse adipocytes
To determine if hesperetin and naringenin regulate TNF-a-induced lipolysis, we ﬁrst measured FFA levels by performing a fatty
acid assay. 3T3-L1 adipocytes were stimulated with TNF-a in thepresence or absence of hesperetin or naringenin. Hesperetin andnaringenin signiﬁcantly inhibited TNF-a-stimulated FFA secretion(Fig. 1A). To further validate the inhibitory effects of these ﬂavonoids on TNF-a-stimulated FFA secretion, we next performed thefatty acid assay with mouse primary adipocytes. As shown in
Fig. 1B, similar results were obtained with the primary cells. There
were no treatment-related changes in cell viability (Fig. 1C; 3T3-L1
adipocytes; Fig. 1D, primary cells).

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H. Yoshida et al. / Biochemical and Biophysical Research Communications 394 (2010) 728–732

Fig. 1. Hesperetin and naringenin inhibit TNF-a-stimulated FFA secretion frommouse adipocytes. (A) 3T3-L1 adipocytes were pretreated with hesperetin (100 lM)
or naringenin (100 lM) for 30 min and then stimulated with TNF-a (10 ng/mL) for
24 h. FFA levels in the culture medium were measured by fatty acid assay. (B)Mouse primary adipocytes were pretreated with hesperetin (100 lM) or naringenin(100 lM) for 30 min and then stimulated with TNF-a (50 ng/mL) for 24 h, followedby fatty acid assay. (C,D) Cell viability was determined by MTT assay and expressedas the ratio relative to the control (C, 3T3-L1 adipocytes; D, primary cells). Values
are means ± SD of triplicate determinations. *p < 0.01 versus TNF-a-treated cells.
Hes, hesperetin; Nar, naringenin.

3.2. Hesperetin and naringenin suppress TNF-a-stimulated FFA
secretion by inhibiting the NF-jB and ERK pathways
To elucidate the mechanism by which hesperetin and naringenin
inhibit TNF-a-stimulated FFA secretion, we sought to identify the
intracellular signaling pathways that are regulated by hesperetin
and naringenin. Because the NF-jB and ERK pathways are involvedin TNF-a-induced lipolysis [21,22], we ﬁrst determined whether
hesperetin and naringenin directly inﬂuence the TNF-a-induced
activation of the NF-kB and ERK pathways by assessing the inhibitorof jB-a (IjB-a) degradation and ERK phosphorylation. IjB-a forms
a complex with the NF-jB transcription factor, preventing its translocation from the cytoplasm to the nucleus, the site of its transcriptional activity [29]. Thus, IjB-a degradation leads to the activationof NF-jB. As shown in Fig. 2A, IjB-a in adipocytes was degradedafter a 5- to 30-min incubation with TNF-a, and ERK phosphorylation was increased after 10 min of incubation. Hesperetin andnaringenin suppressed the IjB-a degradation and ERK phosphorylation (Fig. 2B and C). To conﬁrm that the NF-jB and ERK pathways are
involved in TNF-a-stimulated FFA secretion, we used MG-132 andU0126, which inhibit the NF-jB and ERK pathways, respectively.
The inhibition of the NF-jB and ERK pathways suppressed TNF-astimulated FFA secretion in 3T3-L1 adipocytes (Fig. 2D). These data
indicate that hesperetin and naringenin suppress TNF-a-induced
adipocyte lipolysis by inhibiting the NF-jB and ERK pathways.
3.3. Hesperetin and naringenin prevent the TNF-a-mediateddownregulation of perilipin and PDE3B
Because the downregulation of antilipolytic genes is a key stepin TNF-a-induced lipolysis [13,14], we next determined whether

Fig. 2. Hesperetin and naringenin suppress TNF-a-stimulated FFA secretion by
inhibiting the NF-jB and ERK pathways. (A–C) Cells were pretreated withhesperetin or naringenin for 30 min and then stimulated with TNF-a for 5–
30 min. Total cell lysates were extracted, and IjB-a degradation and ERK
phosphorylation were analyzed by Western blotting. b-Actin served as the loading
control. Experiments were repeated three times. (D) Cells were pretreated with
MG-132 (10 lM) or U0126 (10 lM) for 30 min, and then stimulated with TNF-a(10 ng/mL) for 24 h. FFA levels were measured by fatty acid assay. Values aremeans ± SD of triplicate determinations. *p < 0.01 versus TNF-a-treated cells. MG,
MG-132; U, U0126.

hesperetin and naringenin inﬂuence the transcription of perilipin
and PDE3B. Consistent with previous reports, TNF-a downregulated perilipin and PDE3B mRNA (Fig. 3A and B). However, hesperetin and naringenin prevented the TNF-a-induced decrease inperilipin and PDE3B mRNA. We next investigated whether the
NF-jB and ERK pathways are involved in the TNF-a-mediateddownregulation of perilipin and PDE3B by assessing the effects of
MG-132 and U0126. Interestingly, the inhibition of the ERK pathway, but not the NF-jB pathway, blocked TNF-a-mediated downregulation of perilipin and PDE3B mRNA (Fig. 3C and D). These
results indicate that hesperetin and naringenin prevent TNF-amediated downregulation of perilipin and PDE3B through the inhibition of the ERK pathway.
3.4. Hesperetin and naringenin inhibit TNF-a-stimulated FFA secretion
through the downregulation of IL-6
Although we determined that the inhibition of the ERK pathway
by hesperetin and naringenin is required for the recovery of antilipolytic gene expression, it was unclear how the inhibition of
the NF-jB pathway by these ﬂavonoids suppressed the TNF-astimulated FFA secretion. TNF-a is a powerful local regulator within adipose tissue and plays a pivotal role in the production of otheradipokines [2,30]. Thus, it is possible that TNF-a produces otheradipokines through the activation of the NF-jB pathway that lead
to FFA secretion in an autocrine manner. One candidate adipokine
is interleukin-6 (IL-6), which is also involved in insulin resistanceand adipocyte lipolysis [31,32]. Therefore, we examined whether
hesperetin and naringenin inﬂuence TNF-a-induced IL-6 synthesis.
As shown in Fig. 4A, hesperetin and naringenin signiﬁcantly downregulated the TNF-a-induced transcription of IL-6. We then used
MG-132 and U0126 to determine if the NF-jB and ERK pathways
are involved in TNF-a-induced IL-6 synthesis. Inhibition of the
NF-jB pathway, but not the ERK pathway, blocked the TNF-a-induced transcription of IL-6 (Fig. 4B). These results suggest that hes-

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Fig. 3. Hesperetin and naringenin prevent TNF-a-mediated downregulation of
perilipin and PDE3B. (A–D) Cells were pretreated with hesperetin, naringenin, MG132 or U0126 for 30 min and then stimulated with TNF-a for 3 h. Total RNA wasextracted to measure the mRNA levels of perilipin and PDE3B by real-time RT-PCR.
Results are the means ± SD of three independent experiments. #p < 0.01 versus
control cells. Hes, hesperetin; Nar, naringenin; MG, MG-132; U, U0126.

peretin and naringenin inhibit TNF-a-induced IL-6 synthesis
through the inhibition of the NF-jB pathway. To conﬁrm that IL6 induces adipocyte lipolysis, we assessed the FFA secretion. As
shown in Fig. 4C, IL-6 stimulated FFA secretion from 3T3-L1 adipocytes, although the effect was weaker than that of TNF-a. Cotreatment with TNF-a and IL-6 additively increased FFA secretion. To
determine if the TNF-a-induced IL-6 production promotes adipocyte lipolysis in an autocrine manner, we assessed the effects of
IL-6 neutralizing antibody on TNF-a-stimulated FFA secretion.
Neutralization of endogenous IL-6 suppressed the TNF-a-stimulated FFA secretion (Fig. 4D). Collectively, these data indicate thatthe inhibition of the NF-jB pathway by hesperetin and naringenin
suppresses TNF-a-stimulated FFA secretion through the downregulation of IL-6.
4. Discussion
In this report, we demonstrated that hesperetin and naringenin
inhibit TNF-a-induced adipocyte lipolysis (Fig. 4E). We showed forthe ﬁrst time that these ﬂavonoids inhibit both the NF-jB and ERK
pathways, which in turn suppress TNF-a-stimulated FFA secretion.
The inhibition of the ERK pathway is required for the recovery of
perilipin and PDE3B mRNA. In contrast, the inhibition of NF-jB is
involved in the downregulation of IL-6, which promotes FFA secretion in an autocrine manner. Thus, this study reveals a mechanism
by which hesperetin and naringenin inhibit TNF-a-induced adipocyte lipolysis.
It is particularly interesting that hesperetin and naringenin regulate adipocyte function. Although previous studies showed that
hesperetin and naringenin have various pharmacological properties including anti-inﬂammatory activity, most of these studies
used non-fat cells, such as macrophages [26,27]. Despite recent
studies demonstrating that adipocytes secrete bioactive factors in-

Fig. 4. Hesperetin and naringenin inhibit TNF-a-stimulated FFA secretion throughthe downregulation of IL-6. (A,B) Cells were pretreated with hesperetin, naringenin,
MG-132 or U0126 for 30 min and then stimulated with TNF-a for 1 h. Total RNA
was extracted to measure the mRNA levels of IL-6 by real-time RT-PCR. Results are
the means ± SD of three independent experiments. *p < 0.01 versus TNF-a-treatedcells. (C) Cells were stimulated with TNF-a (10 ng/mL) and/or IL-6 (100 ng/mL) for
24 h. FFA levels were measured by fatty acid assay. Values are means ± SD of
triplicate determinations. #p < 0.01 versus control cells. (D) Cells were pretreatedwith control IgG (10 lg/mL) or IL-6 neutralizing antibody (10 lg/mL) for 30 min
and then stimulated with TNF-a for 24 h. FFA levels were measured by fatty acidassay. Values are means ± SD of triplicate determinations. *p < 0.01 versus TNF-atreated cells. (E) Schematic representation of the inhibition of TNF-a-induced
lipolysis by hesperetin and naringenin. Hes, hesperetin; Nar, naringenin; MG, MG132; U, U0126.

volved in inﬂammation and metabolic disorders [2], the effects ofhesperetin and naringenin on adipocyte function remain largely
unknown. In this study, we demonstrated that hesperetin andnaringenin inhibit TNF-a-induced adipocyte lipolysis through theregulation of ERK-dependent antilipolytic gene expression and
NF-jB-dependent IL-6 synthesis. Thus, hesperetin and naringenin
may prevent obesity-induced insulin resistance by inhibiting
TNF-a-stimulated FFA secretion from adipocytes.
Understanding the primary target molecules of hesperetin andnaringenin is key to the evaluation of these ﬂavonoids as preventive or therapeutic agents for metabolic disorders. There is accumulating evidence that ﬂavonoids, including hesperetin andnaringenin, modulate the activity of a number of protein kinases
that regulate various intracellular signaling cascades, such as phosphoinositide 3-kinase (PI3K), Akt/protein kinase B (Akt/PKB), tyrosine kinases, protein kinase C (PKC) and mitogen-activated protein
kinase (MAPK). This modulatory action is mediated via the bindingof the ﬂavonoids to the ATP-binding sites on enzymes ([33] andreferences therein). In addition to protein kinases, the proteasomeis also a target for ﬂavonoids. Several studies have shown thatﬂavonoids such as apigenin and quercetin inhibit the chymotryp-

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H. Yoshida et al. / Biochemical and Biophysical Research Communications 394 (2010) 728–732

sin-like activity of the proteasome [34,35]. In this study, we showthat hesperetin and naringenin suppress TNF-a-induced ERK phosphorylation and IjB-a degradation. The inhibition of ERK phosphorylation by these ﬂavonoids may result from modulating
upstream regulatory kinases. In contrast, the inhibition of IjB-a
degradation may result from inhibiting proteasome activity. Thus,
hesperetin and naringenin are likely to have extensive target molecules and regulate cellular function.
Another important ﬁnding in our study is that hesperetin andnaringenin regulate IL-6 synthesis in adipocytes. The expressionof IL-6 and TNF-a is elevated in obesity [36]. In addition, IL-6 impairs insulin signaling and promotes insulin resistance [32,37].
Thus, the control of IL-6 expression is an important strategy forthe improvement of insulin resistance. This study shows that hesperetin and naringenin inhibit TNF-a-induced IL-6 synthesis. Thisﬁnding suggests that hesperetin and naringenin exert inhibitory
effects on IL-6-mediated metabolic disorders.
5. Conclusion
This data suggest that hesperetin and naringenin inhibit TNF-ainduced adipocyte lipolysis and that they may be useful for ameliorating insulin resistance. Future studies will verify the ability ofhesperetin and naringenin to inhibit lipolysis-related disorders
in vivo.
Acknowledgments
The authors thank Prof. Satoru Takahashi, Prof. Toshikazu Matsuoka and Ms. Nao Setoguchi (Kyushu University of Health andWelfare) for their kind support and helpful suggestions. This work
was supported in part by a Grant-in-Aid for Scientiﬁc Research(#20880032; to H. Yoshida) from the Japan Society for the Promotion of Science.
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